A Universal Protein Tag for Delivery of SiRNA-Aptamer Chimeras

Liu, Hong Yan; Gao, Xiaohu

Expression and characterization of dsBRD-His 18 protein tag. To add endosomal escape capability, a short polyhistidine peptide was added to dsRBD. The dsRBD domain comes from the first 172 amino acids of human protein kinase R (hPKR), and has two double-strand RNA binding motifs (dsRBM1 and dsRBM2) for cooperative and dsRNA-specific binding 31 . Because dsRBM1 towards the N terminal dominates the binding with dsRNA 32 , we introduced the histidine peptide towards the C terminal ( Figure 1 ) to minimize impact on dsRBD's biological activity. In theory, the endosomal escape capability should increase with longer His chain; on the other hand, long His chain could potentially interfere with dsRBD protein folding and binding. To achieve a balance, dsRBD with Cterminal Histidines of various lengths (His n , n 5 0, 12, 18, and 24) were cloned into the PET28a (1) vector. BamH1 and Xho1 restriction enzyme sites were introduced to the 59-and 39-flanking region by PCR, respectively. Because all the genetic constructs contain His 6 at the N-terminal from the cloning vector (this Nterminal His 6 has been previously proved to have no impact on dsRBD binding) 26 , the total numbers of His encoded by the final constructs are 6, 18, 24, and 30, respectively (sequences see Methods).